There has been a rapid and steady progress in the ability to clone and recombine DNA molecules in recent years. These advances began with the discovery of restriction enzymes that were capable of cleaving double-stranded DNA, so that DNA fragments were produced that could be recombined to generate new recombinant molecules. The revolution was extended by the discovery and development of the polymerase chain reaction (PCR), which allowed rapid amplification of particular DNA segments, producing large amounts of material that could be subsequently cleaved and recombined with other DNA molecules.
Despite the power of these digestion and amplification techniques, however, there remains substantial room for improvement. Restriction enzymes are expensive and sometimes inefficient or available in crude, contaminated preparations. Further, PCR amplification often yields products that are refractory to direct cloning, due to addition of a terminal 3′-dAMP residue by many thermophilic DNA polymerases, including Taq, the most commonly-used enzyme for PCR.
Particularly desirable systems would generate hybrid products suitable for nucleic acid recombination with minimal reliance on restriction enzymes, would provide for efficient recombination, and would be generally useful for recombination between nucleic acids having a wide variety of chemical structures.
Jarrell et al, see WO 00/40715 and U.S. Pat. No. 6,358,712, teach methods for library assembly involving generation of double stranded products with 3′ or 5′ overhangs. However, this method uses polymerase chain reaction, thus requiring thermal cycling and exponential amplification. Furthermore, Jarrell discloses use of a double stranded DNA as template.
Methods using production of recombined products include molecular evolution and “DNA shuffling”. Molecular evolution is a powerful tool for producing novel proteins with enhanced or unique selectable properties. Proteins with enhanced enzymatic activity, enhanced thermal stability, or enhanced stability in organic solvents or defined media such as alkaline or high salt media, novel enzymatic activity, or other desired features may be produced by recombining pieces of mutagenized variants of a single gene or pieces of different genes to form hybrid proteins. In general, in vitro directed evolution involves generation of hybrid polynucleotides, amplification of the polynucleotides, and screening to select a protein having the desired property or properties. “DNA shuffling” involves mutagenesis of a single gene rather than reassembly of multiple domains from related genes. Known methods for DNA shuffling include mutagenesis of a gene, selection of mutants, fragmentation by DNase I, and recombination of the fragments in vitro using PCR (Stemmer, Proc. Natl. Acad. Sci. 91:10747–51 (1994); Stemmer, Nature 370:389–91 (1994)).
Analyses have revealed that many proteins are composed of a number of discrete domains. The individual domains of such a protein are often involved in specific functions that contribute to the protein's overall activity. A number of domains have been found to be evolutionarily mobile. Mobile domains are characterized by their ability to fold independently. “Mosaic proteins” that include multiple mobile domains have been characterized and appear to have arisen through evolution by “exon shuffling.” The natural process of exon shuffling can be mimicked in vitro by generating and screening libraries of exon shuffled genes (Kolkman and Stemmer, Nature Biotechnology 19:423–28 (2001)). The domain-encoding exons in such libraries may be from closely related or divergent genes. Further, domain-encoding exons may be derived from related genes from the same species or homologs from closely-related species.
Thus, there is a serious need for methods of generating double stranded nucleic acid sequences comprising at least one defined single stranded DNA portion, where the double stranded nucleic acid sequences are generated from RNA using methods which do not require and thus avoid thermocycling, and where the double stranded nucleic acid sequences can be random, can be selected from a pool of all mRNA sequences, or can be selected from a related sequences (e.g., all sequences encoding a particular domain, sequences encoding members of a superfamily of genes, and the like).
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.